Synthesis, structures, molecular docking, cytotoxicity and bioimaging studies of two novel Zn(II) complexes

Article abstract of DOI:10.1016/j.ejmech.2016.05.013

Two novel compounds [Zn₂(Endc)₂(bipy)₂(H₂O)₃]·4(H₂O)·2(O)(1), [Zn₂(Endc)₂(phen)₂(H₂O)]·(O)(2) (bipy Combining double low line 2,2-bipyridine, phen Combining double low line 1,10-phenanthroline, and Endc Combining double low line endo-norbornene-cis-5,6-dicarboxylicacid) have been synthesized and characterized. In this paper abbreviations are FS-DNA (fish sperm DNA), HeLa (human cervix epithelia carcinoma cells), KB (human oral epithelial carcinoma cells), LO₂ (human liver cell L-O₂), EtBr (ethidium bromide), DMF (Dimethyl Formamide), MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]). The binding of complexes with Fish Sperm DNA were measured by electronic absorption spectra and fluorescence spectroscopy. The ability of these complexes to cleave the pBR322 plasmid DNA or the KB and HeLa DNA extracted in our laboratory was demonstrated by gel electrophoresis assay. The cytotoxic effects of these complexes were examined on two tumor cell lines, HeLa, KBr and one normal cell line LO-2. UV absorption and fluorescence spectra indicate the ability of the complexes bond to DNA with different binding affinity. Gel electrophoresis assay demonstrates which one complex more effective DNA-cleavage activity. The cytotoxic activity of the complexes was tested against two different cancer and one normal cell lines. The two complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate and lower cytotoxicity toward the normal cell lines. The unique interaction mode with DNA and cancer cells inhibition effect clearly revealed the relationship between the structure and the activity of the novel antitumor agent Zn(II) complexes.

Full text of DOI:10.1016/j.ejmech.2016.05.013

Accepted Manuscript
Synthesis, structures, molecular docking, cytotoxicity and bioimaging studies of two
novel Zn(II) complexes
Enjun Gao, Na Sun, Shaozhong Zhang, Yuqing Ding, Xue Qiu, Yang Zhan,
Mingchang Zhu
PII:
S0223-5234(16)30390-7
10.1016/j.ejmech.2016.05.013
EJMECH 8602
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2016
Revised Date: 11 April 2016
Accepted Date: 5 May 2016
Please cite this article as: E. Gao, N. Sun, S. Zhang, Y. Ding, X. Qiu, Y. Zhan, M. Zhu, Synthesis,
structures, molecular docking, cytotoxicity and bioimaging studies of two novel Zn(II) complexes,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.013.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Confocal fluorescence images of KB cells stained with complex 1and complex 2.

ACCEPTED MANUSCRIPT
Synthesis, structures, molecular dockingꢀcytotoxicity and
bioimaging studies of Two novel Zn(II) complexes
EnjunGao*, Na Sun, Shaozhong Zhang, Yuqing Ding,
XueQiu, Yang Zhan, Mingchang Zhu*
The key Laboratory of the Inorganic Molecule-Based Chemistry of Liaoning Province and Laboratory of
Coordination Chemistry, Shenyang University of Chemical Technology, Shenyang 110142,PR China
Abstract
Two
novel
compounds
[Zn2(Endc)2(bipy)2(H2O)3]·4(H2O)·2(O)(1),
[Zn2(Endc)2(phen)2(H2O)]·(O)(2) (bipy= 2,2-bipyridine, phen = 1,10-phenanthroline,
and Endc = endo-norbornene-cis-5,6-dicarboxylicacid) have been synthesized and
characterized. In this paper abbreviations are FS-DNA (fish sperm DNA), HeLa
(human cervix epithelia carcinoma cells), KB (human oral epithelial carcinoma cells),
LO2 (human liver cell L-O2), EtBr (ethidium bromide), DMF (Dimethyl Formamide),
MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]). The binding of
complexes with Fish Sperm DNA were measured by electronic absorption spectra and
fluorescence spectroscopy. The ability of these complexes to cleave the pBR322
plasmid DNA or the KB and HeLa DNA extracted in our laboratory was
demonstrated by gel electrophoresis assay. The cytotoxic effects of these complexes
were examined on two tumor cell lines, HeLa, KBr and one normal cell line LO-2.
UV absorption and fluorescence spectra indicate the ability of the complexes bond to
DNA with different binding affinity. Gel electrophoresis assay demonstrates which
one complex more effective DNA-cleavage activity. The cytotoxic activity of the
complexes was tested against two different cancer and one normal cell lines. The two
complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate
and lower cytotoxicity toward the normal cell lines. The unique interaction mode with
DNA and cancer cells inhibition effect clearly revealed the relationship between the
structure and the activity of the novel antitumor agent Zn(II) complexes.
Keywords: HeLa and KB cells; DNA binding; Apoptosis; Cytotoxicity
* Corresponding author at: Department of Coordination Chemistry, Shenyang University
of Chemical Technology, Shenyang 110142, China. Tel.: +86 24 89385016; fax: +86 24
89388211.
E-mail: enjungao@163.com(E.J. Gao)or mczhu@syuct.edu.cn(M.C. Zhu)

ACCEPTED MANUSCRIPT
1. Introduction
For many years, there is some evidence demonstrated that other biological targets
may be important in anticancer mechanisms, it is generally accepted that DNA is the
primary biological drug target [1]. So farꢀthe DNA or RNA interactions with transition
metal complexes have been researched for the development of anticancer drugs and
effective chemotherapeutic agents for cure numerous diseases[2]. In our best
knowledge, in this regard, cisplatin (cis-[PtCl2(NH3)2] is one of the most cogent
anti-tumor drugs available for therapeutic of solid tumors[3], particularly, germ cell
tumors, ovarian, lung, head and neck, bladder cancers and so on [4]. It is received that
to mediate its cytotoxic mechanism of action by forming adducts with DNA, which
are believed to be capable of disrupting replication and transcription,[5, 6] Thereby can
prevent cancer exponentially. The great success in the clinical treatment of human
malignancies has motivated researches in the area of inorganic antitumor agents.
However, the side effect of cisplatin is so sharp that individuals often can’t stand it is
pain and give up treatment. Taking these findings into account, we referring to a large
number of related literature, doing a thorough research. Finally selected by chelating
agent ligands: 2,2-bipyridine (bipy) and 1,10-phenanthroline (phen).
1,10-Phenanthroline has been extensively used for decades as a chemically versatile
module exhibiting a fortunate combination of structural and chemical properties[7].
Another important property of 1,10-phenanthroline is connected to its stronger
hydrophobic interaction and larger plate area, that prompts intercalation or groove
binding with DNA or RNA in the metal complexes[8]. In contrast to phen ligands, the
2,2′-bipyridine ligand has been extensively used as a metal chelating ligand due to its
robust redox stability and ease of functionalization.[9, 10] This kind of reagents have
been widely employed and get many benefits, especially in combination with different
transition metals, finding applications in therapeutics as DNA cleaving agents and
tracking DNA footprint tool and so on [11-13]. Used of chelating agents has played an
important role in bioinorganic chemistry [2].
On the other hand, Pt(II), is a very rare and expensive metal, therefore, through
consulting and studying, we use Zn(II) instead of Pt(II). Zinc(II), a ubiquitous ion, is

ACCEPTED MANUSCRIPT
essential for all life forms, has established its importance in biology through both
structural and activity[14]. It plays an important role in various biological system, it is
critical for numerous cell processes and is a major regulatory ion in the metabolism of
cell [15-17]. Among non-platinum metal ions, the Zn (II) ion has the potential to
influence many aspects of cellular signal through its effect on zinc-binding proteins,
because there are many transcription factors and enzymes containing zinc-binding
sites. [18, 19] It is also well known that Zn(II) is useful for reducing the cardio- and heap
toxicity induced by some anticancer drugs[20]. However, little data on the cytotoxicity
of zinc-based compounds against human cancer cell lines are known.[21, 22] Also, some
Zn(II) complexes, which strongly bind and cleave DNA[23, 24], exhibit prominent
anticancer activities and regulate apoptosis.[25]
As a consequence, we select Zn (II), Endc (one our own synthetic ligands), bipy and
phen cultivate metal complexes. As a part of our interest in the experimental process,
characterization and anticancer activities of Zn(II) complexes containing Endc-based
ligand. In the present paper, we report the preparation, structures and DNA binding
properties of two new Zn (II) complexes chelated by L (L1=2,2-bipyridine, L2
=1,10-Phenanthroline) ligands, namely [Zn2(Endc)2(bipy)2(H2O)3]·4(H2O)·2(O) (1)
and [Zn2(Endc)2(phen)2(H2O)]·(O) (2). The structures of both complexes were
identified by elemental analysis, IR and single crystal X-ray technique.
2. Experimental section
2.1 Materials and Physical measurements
All chemicals and reagents purchased were of reagent grade and used without further
purification unless otherwise noted and distilled water were used. Fish Sperm DNA
and pBR322 plasmid DNA were purchased from China. HeLa and KB cell DNA
implemented here were extracted by us Elemental analysis (C, H and N) was
performed on a model Finnigan EA 1112. The IR spectra were obtained as KBr
pellets on a Nicolet FT-IR 470 spectrometer. UV-visible (UV-Vis) spectra were
recorded on a Shimadzu UV-240. Fluorescence measurements were carried out on a
PerkinElmer LS55 flourescence spectro fluorometer.
2.2 Syntheses

ACCEPTED MANUSCRIPT
2.2.1 Syntheses of endo-norbornene-cis–5,6-dicarboxylicacid.
Carbic anhydride was synthesized by the reaction of maleic anhydride with cyclo
peantadiene in a tri-flask at 160 to 190°C until no back heat flow. The reaction was a
cyclo addition to abide by the Diels-Alder rule, along with the performing of the
reaction, there was a transient state intermediate product firstly, then transforming into
carbic anhydride.[26] It was going to a get a white precipitation after putting the
mixture into diethyl ether for dissolving at room temperature. The powder without
diethyl ether was volatilized and hydrolyzed into Endc in the boiling water for
continuous heating.
2.2.2 Synthesis of the complexes
[Zn2(Endc)2(bipy)2(H2O)3]·4 (H2O)·2(O) (1):An aqueous solution (10ml) containing
0.1 mmol of Endc and 10 ml of ethanol involving 0.1 mmol of Bipy was mixed, and
then 0.1 mmol of Zn(NO3)2•6H2O in 10 ml of water solution was added into the
above mixture. The resultant solution was stirred, and the pH value was adjusted to
6.37 with 0.5ml/L KOH. The solution was continuously stirred for 5 h until the pH
value was constant at 5.73. The mixture was condensed with rotatory evaporation, and
25 ml of a white clear concentrated solution obtained was kept at room temperature.
Slightly white transparent crystals were obtained after a few days.Yield: 60%. Anal.
calcd (%) for complex C38H54N4O19Zn2(1): C, 45.57; H, 5.43; N, 5.59;O;30.35 Found:
C,45.59; H, 5.44; N, 5.60,O;30.36.IR (cm−1, s, strong; m, medium; w, weak): 3048(b),
1709(m), 1603(m), 1519(s), 1342(s), 1324(s), 1120(s), 983(m), 871(m),
725(m), 617(m), 647(m).
[Zn2(Endc)2(phen)2(H2O)]·(O)(2): The complex (2) also prepared by same
procedure, only bipy was instead of phen. White crystals were produced Yield: 65%.
Anal. calcd (%) for complex C42H38N4O11Zn2(2): C, 55.71; H, 4.23; N, 6.19; O;19.44
Found: C,55.73; H, 4.23; N, 6.20,O;19.45.IR (cm−1, s, strong; m, medium; w,weak):
3059(b), 1716(s),1446(m),1316(s), 1120(s), 1027(s), 982(s), 844(m), 734(m), 617(m).
2.3 X-ray crystal structure determination
Data of complexes (1) and (2) were collected on a Brucker Smart 1000 CCD X-ray
single-crystal diffractometer with Mo Kα radiation (k = 0.71073 Å) at 273 K in the

ACCEPTED MANUSCRIPT
range of 1.44° < h < 26.04° and 2.38° < h < 26.04°. The structure was analysis by
direct methods using the SHELXS-97 program.[27] All non-hydrogen atoms were
refined anisotropic. Hydrogen atoms were located from different Fourier maps.
Crystal data and structure refinement for two Zn (II) complexes are summarized in
Table 1. Selected bond lengths and angles are listed in Table 2. Hydrogen-bond
geometry of two complexes is exhibited in Table 3.
2.4 DNA binding and cleavage
2.4.1 Electronic absorption spectroscopy
UV-visible spectra were recorded on a Shimadzu UV-240. Tris-HCl buffer (pH 7.0)
and a buffer (10 mmol/L) solution of complexes (1) and (2) were titrated with a
concentrated Fish sperm DNA (FS-DNA) solution. The absorbance of the solution at
240 nm, 290 nm, 310nm, and 220 nm, 260 nm, 290 nm was measured initially for
complexes (1) and (2) after each addition of FS-DNA.
2.4.2 Fluorescence spectroscopic studies
For all fluorescence measurements, the entrance and exit slits were maintained at 15
nm, respectively. The sample was excited at 526 nm and its emission observed at 617
nm. The buffer used in the binding studies was 50 mmol/L Trise-HCl pH 7.2,
containing 10mmol/L NaCl. The sample was incubated for 4 h at room temperature
(20 °C) before spectral measurements. Under these conditions, the fluorescence
intensity of the respective complexes, extracted HC-DNA and EtBr was very small
and could be ignore.[28] The interaction of the respective complexes with DNA in vitro
was studied as described in the literature.[29, 30]
2.4.3 Gel electrophoresis experiments
The gel electrophoresis experiments were performed as described earlier Supercoiled
pBR322 DNA (0.33 mg/ml) or as described later the KB and HeLa DNA extraction
by us was reacted with the metal complex in Tris buffer (50 mM Tris-acetate, 18 mM
NaCl buffer, pH 7.2) and the mixtures were incubated for 1.5 h at room temperature.
The samples were electrophoresed for 1.5 h at 90 V on 0.8% agarose gel in
Tris-acetate buffer. After electrophoresis, the gel was stained with 1 mg/ml EtBr and
photographed under UV light.

ACCEPTED MANUSCRIPT
2.5 Cell line and culture
The cell lines used in this experiment were routinely maintained in a RPMI-1640
medium supplemented with 10% (v/v) heat inactivated fetal bovine serum, 2 mmol/L
of glutamine, 100 ꢀg m/L of penicillin, and 100 ꢀg m/L of streptomycin in a highly
humidified atmosphere of 95% air with 5% CO2 at 37 °C.
2.6 Extraction DNA
HeLa and KB cells were grown adherently on the culture flask at 37°C under sterile
conditions after 2–3 days of growth for extraction. Saturated sodium chloride
extraction method was taken to extract DNA.[31]
The cells were washed 2 times with 1 ml PBS (0.1 mol/L, NaCl, Na2HPO4·12H2O,
NaH2PO4·2H2O, pH 7.4) buffer system, then 0.8 ml TE (10 mmol/L Tris–HCl,
1mmol/L EDTA, pH 7.5) was added and the cells were removed by a cell scraper,
which were transferred into a eppendorff tube. The cells specimens were centrifuged
under 10,000 rpm at room temperature. The sediments were suspended in 0.4 ml TE,
digested in 40ꢀl of a buffer containing 20 mg/mL proteinase K and 10% SDs and
incubated overnight at 37°C. A 150ꢀL volume of saturated sodium chloride was
added to the specimen and incubated at 40 °C for 10 min. The mixture was
centrifuged under 10,000 rpm for 10 min to get the supernatant to another tube, in
which 715ꢀl chloroform was added and blended with supernatant, then centrifuged
under 10,000 rpm for 10 min again and the supernatant carefully decanted. An 100ml
volume of absolute ethanol and 1/10 [v/v] of 3 mol/L NaAc pH 5.2 was added, the
solution was mixed for 5 min and then allowed to stand at room temperature for 5 min.
The mixture was centrifuged at 10,000 rpm for 1 min at room temperature, and the
recovered pellet washed with 0.5 ml of 70% ethanol. After removing all traces of
ethanol, the pellet was dissolved in 100ꢀL of TE. A solution of DNA in the buffer
gave a ratio of UV absorbance at 260 and 280 nm of about 1.9:1, indicating that the
DNA is sufficiently free from protein.
2.7 In vitro apoptosis assay
HeLa and KB cells under usual conditions were seeded in a 24-well culture plate at
2× 105 cells per well in a 1 ml culture medium and 24 h later the complexes was

ACCEPTED MANUSCRIPT
added. The concentration of complex was 50ꢀmol/L. The cells were further incubated
for 48 h, and then the level of apoptosis was evaluated in at least three independent
experiments. [32] Annexin-V/Propidium Iodide (PI) binding assay staining was carried
out as described above.
2.8 Cytotoxicity assay
The in vitro cytotoxicity of complexes (1)-(2) was investigated by the MTT assay[33]
on the KB cells and the HeLa cells and LO2 cells, which were grown in 25 cm2 tissue
culture flasks in an incubator at 37°C in a humidified atmosphere consisting of 5%
CO2 and 95% air and maintained in logarithmic growth phase in complete medium
including RPMI 1640, 10% (v/v) heat-inactivated fetal calf serum, 20 mmol/L Hepes,
0.112% bicarbonate, and 2 mmol/L glutamine. The suspensions of cancer cells were
adjusted to cell densities of 5×105 cells/ml in order to ensure exponential growth
throughout drug exposure. Aliquots of 100 ꢀl well of these suspensions were
reattributed into 96-well culture plates and pre-incubate for24h. Then cells were
exposed to the tested complexes of serial concentrations. The complexes were
dissolved in DMF and diluted with RPMI 1640 or DMEM to the required
concentrations prior to use (0.1% DMF final concentration) [34]. After incubation
lasting 72 h, 20 ꢀl of aqueous MTT solution (5 mg/mL) was added to each well and
the cells were incubated continually for another 4 h. The media with MTT were
removed, and 100 ꢀl of DMSO was added to each well in order to dissolve formazan
crystal at room temperature for 30 min. The absorbance of each cell at 450 nm was
determined by analysis with a microplate spectrophotometer. The percentage cell
viability was determined by dividing the average absorbance of each column of
drug-treated wells by the average absorbance of the control wells. Each experiment
was repeated three times, and the discussed IC50 values represent an arithmetic mean
[35-37]
.
2.9. Molecular docking study
The 3D structure of Zn(II) complex were obtained and optimized using Avogadro,[38,
39]
the crystal structure of B-DNA dodecamer was download from PDB(PDB
ID:3JRA). Molecular docking study was performed using a tuned version of

ACCEPTED MANUSCRIPT
AutoDock Vina InYASARA[40, 41]. The grid box was set to overlap the whole DNA
segment and running times of docking were set to 100. The lowest energy
conformation was chosen for further analysis. Chimera was employed to visualize the
docked pose.
3. Results and discussion
3.1 Structure
3.1.1 Structure of[Zn2(Endc)2(bipy)2(H2O)3]·4 (H2O) ·2(O)
A single-crystal X-ray diffraction analysis revealed that compound (1) crystallizes in
the Triclinic space group P-1(Fig.1).Compound (1) is an extremely rare example of a
1D spiral-like network. As shown in Fig.2, the structure of (1) contains tow Zn(II)
cations, two bipy ligands, two Endc ligands, three water molecules, and four
uncoordinated water molecules and two dissociative oxygen atoms. The coordination
environment of Zn1 is a distorted ZnO4N2 octahedral geometry Fig.S1(a), with two
nitrogen donors from the bipy ligands and three oxygen donors from
intra-molecularly water molecules, another oxygen donor from the Endc ligands.
Whereas the apical positions are occupied by O5 and O7 from two different
intra-molecularly water molecules with an O5−Zn−O7 bond angle of 175.85°. As
shown in Fig.S2, two bipy ligands formed π−π stacking. For convenience (Fig. S3),
the bipy ligands containing N1 and N2 are designated bip-A and bip-B, respectively.
3.1.2 Structure of [Zn2(Endc)2(phen)2(H2O)]· (O)
The asymmetric unit of (2) consists of two crystal graphically independent Zn(II) ions,
two phen ligand, and two Endc ligands (Fig.3).The Zn atoms are coordinated with two
oxygen atoms from one Endc ligands and one intra-molecularly water molecules and
two nitrogen atoms from one phen ligand in the form of distorted tetrahedral
geometry(Fig.4). As shown in Fig.S1 (b), the Zn distances are in the range of
2.012−2.188 Å, which are in the normal range according to previously reported Zn
(II)-based carboxylates. As shown in Fig.S4 and S5, the most fascinating structural
feature of (2) is that the infinite double-stranded helix and the architecture are
composed of two equivalent frameworks.
3.2 UV absorption

ACCEPTED MANUSCRIPT
Electronic absorption spectroscopy is one of the most important techniques to
examine the binding mode of DNA with metal complexes.[42, 43] If the binding mode is
intercalation, then the intercalated ligand’s π orbital can couple with the π orbital of
the DNA base pairs, thus, the π-π* transition energy is decreasing and resulting in the
bath ochromism.[44] On the other hand, the coupling π orbital is partially filled by
electrons, thus, decreasing the transition probabilities and concomitantly resulting in
hypochromism[44]. This result suggested that the mode of the complex binding to
FS-DNA involves a strong stacking interaction between an aromatic group and the
base pairs of DNA. It seems to be generally accepted that the extent of the
hypochromism in the UV band is consistent with the strength of intercalative
interaction [45, 46]. So, in order to provide evidence for the possibility of binding of
each complex to FS-DNA, spectroscopic titration of a solution of the complexes with
FS-DNA has been performed. As shown in Fig.5 and 6, when addition of an
increasing amount of FS-DNA to the complexes, the hypochromism and a slight red
shift were observed, this indicates moderate binding of the complexes with DNA. The
similar hypochromism was previously observed for other complexes [47, 48]. The
spectral changes are characterized by one or more isosbestic points (complex (1):
240nm, 290nm, 310nm; complex (2): 220nmꢀ260 nm,290nm) well maintained till the
end of the titrations of the complexes with DNA, thus we can eliminate the presence
of species other than the free and the intercalated complexes, which can be assigned
to the π-π* (at 220, 240nm) and n-π* (at 260, 290 and 310nm) transitions of the
2,2'-Bipyridine and 1,10-phenanthroline rings. Such an observation is the result of the
so-called ‘additive effect’ of absorption spectra which the metal atoms are
simultaneously coordinated to two ligands [49], which are H2Endc ligands and H2Bipy
(for(1)) or Phen ligands (for (2)).
With increasing concentrations of DNA added, significant hyperchromism, red-shift
and blue-shift are observed. There are 93% hyperchromism with a 9-nm red-shift
(from 240 to 220nm) at 220nm and 40% hyperchromism with an 8-nm blue-shift
(from 295 to 267nm) at 290nm for (2). Different from the peak absorption intensities
of compound (2), the peak absorption intensities of compound (1) increase about 8%

ACCEPTED MANUSCRIPT
at 290nm and do hardly change at 310nm, which may be due to the coordination
between 2,2′-bipyridyl moiety and Zn2+.
The presence of the isobestic points indicates that there is equilibrium between DNA
bound and free form of the metal complex and complex (2) better than (1).
3.3 Fluorescence spectrum
Fluorescence quenching measurements can be used to monitor metal binding [50]. EtBr
is a conjugate planar molecule. Its fluorescence intensity is very weak, but it is greatly
increased when EtBr is specifically intercalated into the base pairs of double-stranded.
It has been reported that the enhanced fluorescence could be quenched by the addition
of another molecule [51, 52]. The addition of the complex to extracted HC-DNA
pretreated with EtBr causes an appreciable reduction in fluorescence intensity,
showing that the Zn (II) complexes compete with EtBr to bind with extracted
HC-DNA. According to the classical SterneVolmer equation[53]: I/I0 = 1+ Ksq r, where
I and I0 represent the fluorescence intensities in the absence and presence of the
complex, respectively, and r is the concentration ratio of the complex to DNA, Ksq is
a linear SterneVolmer quenching constant dependent on the ratio of the bound
concentration of EtBr to the concentration of DNA. The Ksq value is obtained as the
slope of I/I0 versus r linear plot. The fluorescence quenching curves of DNA-bound
EtBr by the two complexes are given in Fig. 7 and 8. The quenching curves illustrate
that the complexes bind to DNA in the order of Ksq(2)>Ksq(1). The data also shows
that the intercalary ability of the coordinated ligands varies as phen>bipyinthe two
complexes. Obviously, the result follows the order of complex (2) > complex (1).
3.4 Cleavage of extracted cell DNA
Gel electrophoresis is used in molecular biology, genetics, microbiology and
biochemistry.[54, 55] In order to investigate the nuclease activity of complexes (1) and
(2), agarose gel electrophoresis was carried out following the incubation of these with
super coiled pBR322 DNA in 50 mmol/L Tris–HCl50 mmol/L NaCl buffer (pH 7.2).
The DNA cleavage behavior of complexes was analyzed by monitoring the
conversion of the supercoiled DNA form (Form I) to the nicked (Form II) and linear
forms (Form III). The gel picture displaying the DNA molecules is shown in Fig. 9. It

ACCEPTED MANUSCRIPT
is well known that circular plasmid DNA migrates faster than that of its linear form in
electrophoresis due to closed circular conformations [56]. If one strand of DNA is
nicked, the supercoil will relax and a slower moving conformation will be produced.
Complexes (1) and (2) effectively cleave double stranded DNA even in the absence of
any additional external agents and cleavage efficiency of complexes remarkably
increases with higher concentrations. Furthermore, the DNA of pBR322 plasmid
effect on complex (1) and (2). Complex (2) complete conversion of supercoiled form
into the nicked form was observed with by Comparison and analysis results of
cleavage the two complex.
3.5 Apoptotic study
The mode of cell death was studied by Annexin-V/ Propidium Iodide (PI) binding
assay with complex (1) and (2) in KB cells. Respectively, In 6 h and 12 h incubation
of the cells with (1) and (2) and subsequent photo irradiation (400–700 nm, 10 cm–2)
caused about 90% of the cells to undergo apoptosis, They were shown in Fig.10 and
11. The figures suggest which indicates that the number of the dead cancer cells
increases gradually as the time large in early apoptotic (lower right quadrant) or late
apoptotic/necrotic (upper right quadrant). Morphological features of apoptotic and
viable KB cells stained with Annexin V-FITC and Propidium Iodide (PI), the
experiment is repeated by reproducing the conditions as nearly as possible, under
confocal fluorescence images of KB cells, the result is depicted in Fig. 12 and 13.
Compared (1) with (2), the results suggest that the complex (2) better than complex (1)
in terms of cancer cell apoptosis.
3.6 Cytotoxicity assay
MTT assays were used to evaluate the anticancer activity of Zn 1 and Zn 2. Two
human tumor cell lines, HeLa, KB and a normal cell line LO-2, were tested (Table 4).
For comparison purposes, cisplatin comparison was also assayed under the same
experimental conditions.[57, 58] The IC50 values for all complexes were comparable to
that of cisplatin. The introduction of phenyl in the bipy ligand resulted in a slight
increase in the anticancer activity with IC50 values ranging from 20–40ꢀmol/L.
Surprising, with regard to the triphenyl substituted complex (2), a significantly

ACCEPTED MANUSCRIPT
enhanced anticancer activity was observed. The IC50 values of complex (2) toward
cancer cells were similar to the cisplatin. This result is similar to other Zn compound
documents.[59, 60] The greater activity of both complexes (1) and (2) confirms that the
phenyl on the ligand has an important impact on the cytotoxicity of these complexes.
Additionally, all of the Zn (II) complexes exhibited lower cytotoxicity toward the
normal cell line than cisplatin.
3.7 Molecular docking assay
As synthesized Zn(II) complex was docked in to DNA protein in order to predict the
possible binding mode of Zn(II) complex with DNA.[61] The result showed that Zn(II)
complex could bind the DNA molecule through hydrogen bonds. In Fig.14 shows
oxygen and nitrogen atoms chelated with Zn(II) forming hydrogen bonds. The
compound that only contain ball-model is The Zn(II) complex molecular, the others is
DNA protein in Fig.14. It was a significantly preference of Zn(II) complex that bind
to the minor groove of DNA protein. It had been proved that most of the synthesized
metallic complexes bound the minor groove inside the DNA protein,[62, 63] which
would affect the stability of DNA, thus exhibiting DNA cleavage capacity.
4. Conclusion
In this paper, we designed and synthesized a series of two novel organic-metal Zn(II)
complexes based on a simple structure–COOH, where ancillary -COOH ligands with
increasing conjugation intensity, namely endo-norbornene-cis–5,6-dicarboxylic acid,
were synthesized. Herein, we explored the biochemical and potential anti-tumor
activities of complexes with different DNA in different assay. Comparative studies
show that emission intensity, quantum efficiency, as well as cell physiology relevant
parameters, including cell uptake efficiency, cytotoxicity and binding protein of
complex (2) are much better than those of complex (1), which shows that the structure
of complexes affect cell viability and efficient apoptosis.
Acknowledgments
We gratefully acknowledge the Natural Science Foundation of China (No. 21171118)
and the Distinguished Professor Project of Liaoning province.
Appendix A. Supplementary material

ACCEPTED MANUSCRIPT
The coordination geometry structure, 2d and 3d frame work of two compounds
contain the supplementary crystallographic data for this paper.

ACCEPTED MANUSCRIPT
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Sun, Y.G., et al., Synthesis, characterization, and interaction with DNA of Cu(II) and Zn(II)
complexes with 2,2-bipyridyl-6,6-dicarboxylic acid. Journal of Coordination Chemistry, 2013.
66(14): p. 2455-2464.
Zhang, N., et al., Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient
anticancer drug delivery vehicle. European Journal of Pharmaceutics and Biopharmaceutics,
2015. 94: p. 20-29.
Bugarčić, Ž.D., J. Bogojeski, and R. van Eldik, Kinetics, mechanism and equilibrium studies on
the substitution reactions of Pd(II) in reference to Pt(II) complexes with bio-molecules.
Coordination Chemistry Reviews, 2015. 292(0): p. 91-106.
Srishailam, A., et al., Cellular uptake, cytotoxicity, apoptosis, DNA-binding, photocleavage and
molecular docking studies of ruthenium(II) polypyridyl complexes. J Photochem Photobiol B,
2014. 132: p. 111-23.
Liu, B., et al., Computational design, chemical synthesis, and biological evaluation of a novel
ERK inhibitor (BL-EI001) with apoptosis-inducing mechanisms in breast cancer. Oncotarget,
2015. 6(9): p. 6762-6775.
Zhao, X.S., et al., Synthesis, crystal structures and two-photon absorption properties of
triphenylamine cyanoacetic acid derivative and its organooxotin complexes. Dalton
Transactions, 2015. 44(2): p. 701-709.
Chen, Y.Q., et al., Coordination polymers constructed from 2-(2-thienyl)imidazo 4,5-f
1,10-phenanthroline and isophthalate: synthesis, characterization and luminescent property.
Journal of Coordination Chemistry, 2010. 63(8): p. 1327-1338.
Liu, H., et al., Synthesis, crystal structure, and interaction with DNA and BSA of a chromium(III)
complex with naph-gly Schiff base and 1,10-phenanthroline. Journal of Coordination
Chemistry, 2015. 68(6): p. 1040-1053.
Segura, D.F., et al., Synthesis and biological evaluation of ternary silver compounds bearing
N,N-chelating ligands and thiourea: X-ray structure of [{Ag(bpy)(
μ
-tu)}2](NO3)2
(bpy&#xa0;=&#xa0;2,2
ꢁ-bipyridine; tu&#xa0;=&#xa0;thiourea). Polyhedron, 2014. 79(0): p.
197-206.
10.
11.
12.
Waki, M., et al., A Solid Chelating Ligand: Periodic Mesoporous Organosilica Containing 2,2ꢁ
-Bipyridine within the Pore Walls. Journal of the American Chemical Society, 2014. 136(10): p.
4003-4011.
Bania, K.K. and R.C. Deka, Experimental and Theoretical Evidence for Encapsulation and
Tethering of 1,10-Phenanthroline Complexes of Fe, Cu, and Zn in Zeolite-Y. Journal of Physical
Chemistry C, 2012. 116(27): p. 14295-14310.
Chiniforoshan, H., et al., Pyrazinamide drug interacting with Co(III) and Zn(II) metal ions
based on 2,2 '-bipyridine and 1,10-phenanthroline ligands: Synthesis, studies and crystal
structure, DFT calculations and antibacterial assays. Journal of Molecular Structure, 2015.
1081: p. 237-243.
13.
14.
Yi, Z.H., et al., Hydrothermal syntheses and structural characterizations of organic-inorganic
hybrid materials of the M(II)-ligand/vanadium oxide system (M(II) = Mn(II), Cu(II) and Zn(II);
ligand=2,2 '-bipyridine and 1,10-phenanthroline). Dalton Transactions, 2007(21): p.
2115-2120.
Tan, Y.Q., et al., A new fluorescent probe for Zn2+ with red emission and its application in

ACCEPTED MANUSCRIPT
bioimaging. Dalton Transactions, 2014. 43(21): p. 8048-8053.
15.
16.
Deng, K., Y. Wu, and Q.Y. Chen, BDAZn-GO complex as turn-on selective sensor of BSA. Sensors
and Actuators B-Chemical, 2015. 212: p. 512-516.
Pietrangeli, D., et al., Water-soluble carboranyl-phthalocyanines for BNCT. Synthesis,
characterization, and in vitro tests of the Zn(ii)-nido-carboranyl-hexylthiophthalocyanine.
Dalton Transactions, 2015. 44(24): p. 11021-11028.
17.
18.
Dong, Y.-W., et al., Tunable Luminescence and Application in Dye-Sensitized Solar Cells of
Zn(II)/Hg(II) Complexes: Methyl Substitution-Induced Supramolecular Structures Based on
(E)-N-(6-Methoxypyridin-2-ylmethylene)arylamine Derivatives. Inorganic Chemistry, 2015.
54(16): p. 7742-7752.
Thamilarasan, V., A. Jayamani, and N. Sengottuvelan, Synthesis, molecular structure,
biological properties and molecular docking studies on MnII, CoII and ZnII complexes
containing bipyridine–azide ligands. European Journal of Medicinal Chemistry, 2015. 89(0): p.
266-278.
19.
20.
21.
22.
Chan, K.L., et al., Characterization of the Zn(II) Binding Properties of the Human Wilms’ Tumor
Suppressor Protein C-terminal Zinc Finger Peptide. Inorganic Chemistry, 2014. 53(12): p.
6309-6320.
Qiu, H.J., et al., A novel microwave stimulus remote controlled anticancer drug release system
based on Fe3O4@ZnO@mGd(2)O(3):Eu@P(NIPAm-co-MAA) multifunctional nanocarriers.
Journal of Materials Chemistry B, 2015. 3(34): p. 6919-6927.
Huang, K.-B., et al., Isoquinoline derivatives Zn(II)/Ni(II) complexes: Crystal structures,
cytotoxicity, and their action mechanism. European Journal of Medicinal Chemistry, 2015.
100(0): p. 68-76.
Sherif,
Y.E.
and
N.M.
Hosny,
Anti-rheumatic
potential
of
ethyl
2-(2-cyano-3-mercapto-3-(phenylamino)
acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate and its Co(II), Cu(II) and Zn(II)
complexes. European Journal of Medicinal Chemistry, 2014. 83: p. 338-343.
Zhang, H., et al., Synthesis, crystal structure, cytotoxic activity and DNA-binding properties of
the copper (II) and zinc (II) complexes with 1-[3-(2-pyridyl)pyrazol-1-ylmethyl]naphthalene.
Journal of Inorganic Biochemistry, 2005. 99(5): p. 1119-1125.
23.
24.
25.
Jiang, Q., et al., DNA binding property, nuclease activity and cytotoxicity of Zn(II) complexes of
terpyridine derivatives. Biometals, 2009. 22(2): p. 297-305.
Kaushik, N.K., et al., Synthesis, characterization, and antibacterial and anticancer screening of
{M2+-Co3+-M2+} and {Co3+-M2+} (M is Zn, Cd, Hg) heterometallic complexes. Journal of
Biological Inorganic Chemistry, 2012. 17(8): p. 1217-1230.
26.
27.
28.
Wang, H., et al., Synthesis and properties of a novel Cu(II)-pyridineoxazoline containing
polymeric catalyst for asymmetric Diels-Alder reaction. Rsc Advances, 2015. 5(4): p.
2882-2890.
Jaganyi, D., et al., Kinetic and mechanistic study on the reactions of [Pt(bpma)(H2O)]2+ and
[Pd(bpma)(H2O)]2+ with some nucleophiles. Crystal structure of [Pd(bpma)(py)](ClO4)2.
Dalton Trans, 2006(24): p. 2943-9.
Lv, Y., et al., A series of novel Zn(II) and Mn(II) metal-organic frameworks constructed by
2,4-bis-oxyacetate-benzoic acid: syntheses, structures and photoluminescence. Science
China-Chemistry, 2015. 58(3): p. 448-456.

ACCEPTED MANUSCRIPT
29.
Paitandi, R.P., et al., Interaction of ferrocene appended Ru(II), Rh(III) and Ir(III) dipyrrinato
complexes with DNA/protein, molecular docking and antitumor activity. European Journal of
Medicinal Chemistry, 2014. 84: p. 17-29.
30.
31.
Khan, A.A., et al., Biophysical Interactions of Novel Oleic Acid Conjugate and its Anticancer
Potential in HeLa Cells. Journal of Fluorescence, 2015. 25(3): p. 519-525.
Artyukhin, A.B. and Y.H. Woo, DNA extraction method with improved efficiency and specificity
using DNA methyltransferase and "click" chemistry. Analytical Biochemistry, 2012. 425(2): p.
169-174.
32.
33.
34.
35.
36.
37.
van Rijt, S.H., et al., Potent organometallic osmium compounds induce
mitochondria-mediated apoptosis and S-phase cell cycle arrest in A549 non-small cell lung
cancer cells. Metallomics, 2014. 6(5): p. 1014-1022.
Gao, E.J., et al., Synthesis, crystal structure, interaction with DNA, and cytotoxicity in vitro of a
new mixed ligand-nickel complex: Ni(DBMA)(en)(H2O)(3) center dot 3H(2)O. Journal of
Coordination Chemistry, 2013. 66(17): p. 3004-3013.
Gao, E.-j., et al., Synthesis, characterization, interaction with DNA and cytotoxicity in vitro of
novel pyridine complexes with Zn(II). European Journal of Medicinal Chemistry, 2010. 45(10):
p. 4531-4538.
Zhang, Y., et al., Dual-targeting organometallic ruthenium(ii) anticancer complexes bearing
EGFR-inhibiting 4-anilinoquinazoline ligands. Dalton Transactions, 2015. 44(29): p.
13100-13111.
Sinha, P., et al., Homoleptic bisterpyridyl complexes: Synthesis, characterization, DNA binding,
DNA cleavage and topoisomerase II inhibition activity. Inorganica Chimica Acta, 2015. 432(0):
p. 71-80.
ElHusseiny, A.F., et al., Structure–property relationship studies of copper(I) complexes of
nanosized hypodentate ligands and evaluation of their antitumor and antimicrobial activities.
Journal of Coordination Chemistry, 2014. 68(2): p. 241-260.
38.
39.
MacDonald, C.A. and R.J. Boyd, Molecular docking study of macrocycles as Fk506-binding
protein inhibitors. Journal of Molecular Graphics & Modelling, 2015. 59: p. 117-122.
Parveen, S., F. Arjmand, and I. Ahmad, Enantiomeric in vitro DNA binding, pBR322 DNA
cleavage and molecular docking studies of chiral L- and D-ternary copper(II) complexes of
histidine and picolinic acid. Journal of Photochemistry and Photobiology B-Biology, 2014. 130:
p. 170-178.
40.
41.
42.
43.
Albada, H.B., E. Golub, and I. Willner, Computational docking simulations of a DNA-aptamer
for argininamide and related ligands. Journal of Computer-Aided Molecular Design, 2015.
29(7): p. 643-654.
Mozolewska, M.A., et al., Molecular modeling of the binding modes of the iron-sulfur protein
to the Jac1 co-chaperone from Saccharomyces cerevisiae by all-atom and coarse-grained
approaches. Proteins-Structure Function and Bioinformatics, 2015. 83(8): p. 1414-1426.
Gao, E.J., et al., Synthesis, interaction with double-helical DNA and biological activity of new
Pt(II) and Pd(II) complexes with phenylglycine. Journal of Coordination Chemistry, 2009.
62(21): p. 3425-3437.
Cao, Y.W., et al., Formation and Dissociation of the Interstrand i-Motif by the Sequences
d(XnC4Ym) Monitored with Electrospray Ionization Mass Spectrometry. Journal of the
American Society for Mass Spectrometry, 2015. 26(6): p. 994-1003.

ACCEPTED MANUSCRIPT
44.
45.
Gao, E., et al., Impact of the Carbon Chain Length of Novel Palladium(II) Complexes on
Interaction with DNA and Cytotoxic Activity. Inorganic Chemistry, 2010. 49(7): p. 3261-3270.
Zhu, M.-C., et al., Synthesis, characterization, and DNA interaction of novel Pt(ii) complexes
and their cytotoxicity, apoptosis and molecular docking. RSC Advances, 2015. 5(59): p.
47798-47808.
46.
47.
Ma, C., et al., Cadmium(II) complex with 2-methyl-1H-4,5-imidazoledicarboxylic acid ligand:
synthesis, characterization, and biological activity. Journal of Coordination Chemistry, 2014.
67(21): p. 3551-3564.
Sen, A. and C.E.H. Dessent, Mapping the UV Photophysics of Platinum Metal Complexes
Bound to Nucleobases: Laser Spectroscopy of Isolated Uracil center dot Pt(CN)(4)(2-) and
Uracil center dot Pt(CN)(6)(2-) Complexes. Journal of Physical Chemistry Letters, 2014. 5(19):
p. 3281-3285.
48.
Li, X., et al., Synthesis, Crystal Structure and DNA Interaction of a Novel Three-Nuclear
Cobalt(II) Complex with Schiff Base Derived from 4-Chloroanthranilic Acid and
2,4-Dihydroxybenzaldehyde. Journal of Inorganic and Organometallic Polymers and Materials,
2014. 24(3): p. 582-590.
49.
50.
51.
Kashanian, S., et al., Multi-spectroscopic DNA interaction studies of sunset yellow food
additive. Molecular Biology Reports, 2012. 39(12): p. 10045-10051.
Zhu, M., et al., Synthesis, characterization, and cytotoxicity in vitro of the complex Mn (Hptc)
(phen) (OH) n. Life Sciences, 2012. 90(13-14): p. 519-524.
Cao, G.L., et al., Organic-inorganic heteropoly blue based on Dawson-type molybdosulfate
and organic dye and its characterization and application in electrocatalysis. Electrochimica
Acta, 2013. 106: p. 465-471.
52.
53.
Guan, L., Y. Wang, and H.F. Sheng, Crystal Structure and Fluorescence Property of Dimeric
Complex Mg(phen)(H2O)(4)center dot(2,7-nds)center dot(phen)center dot 4H(2)O (2).
Synthesis and Reactivity in Inorganic Metal-Organic and Nano-Metal Chemistry, 2015. 45(9): p.
1261-1265.
Gao, E.-j., et al., Synthesis and crystal structure of two new dinuclear cobalt(II) complexes
interaction with HeLa cells. European Journal of Medicinal Chemistry, 2011. 46(1): p.
160-167.
54.
55.
Zhu, Z., et al., Simultaneously Sizing and Quantitating Zeptomole-Level DNA at High
Throughput in Free Solution. Chemistry – A European Journal, 2014. 20(43): p. 13945-13950.
Zhou, W., et al., Improving nuclease activity of copper(II)-terpyridine complex through
solubilizing and charge effects of glycine. Journal of Inorganic Biochemistry, 2013. 121: p.
114-120.
56.
57.
Hussein, M.A., et al., Structures, DNA binding, DNA cleavage, and antitumor investigations of
a series of molybdenum(VI) complexes with some N(4) methyl and ethyl thiosemicarbazone
ligands. Journal of Coordination Chemistry, 2014. 67(4): p. 714-727.
Prabhakaran, R., et al., DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase,
NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone
complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography. Journal of
Biological Inorganic Chemistry, 2013. 18(2): p. 233-247.
58.
Wang, Y., et al., Synthesis, characterization and in vitro anticancer activity of the
biomolecule-based coordination complex nanotubes. Journal of Materials Chemistry B, 2015.

ACCEPTED MANUSCRIPT
3(2): p. 296-305.
59.
60.
61.
Pravin, N. and N. Raman, Investigation of in vitro anticancer and DNA strap interactions in live
cells using carboplatin type Cu(II) and Zn(II) metalloinsertors. European Journal of Medicinal
Chemistry, 2014. 85: p. 675-687.
Wang, S.X., et al., Synthesis, characterization and cytotoxicity of Pt(II), Pd(II), Cu(II) and Zn(II)
complexes with 4'-substituted terpyridine. Applied Organometallic Chemistry, 2013. 27(7): p.
373-379.
Icsel, C., et al., New palladium(ii) and platinum(ii) 5,5-diethylbarbiturate complexes with
2-phenylpyridine, 2,2[prime or minute]-bipyridine and 2,2[prime or minute]-dipyridylamine:
synthesis, structures, DNA binding, molecular docking, cellular uptake, antioxidant activity
and cytotoxicity. Dalton Transactions, 2015. 44(15): p. 6880-6895.
62.
63.
Siqueira, R.P., et al., Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition
by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340). Plos One, 2015.
10(8).
Haribabu, J., et al., Synthesis, DNA/protein binding, molecular docking, DNA cleavage and in
vitro anticancer activity of nickel(ii) bis(thiosemicarbazone) complexes. RSC Advances, 2015.
5(57): p. 46031-46049.

ACCEPTED MANUSCRIPT
Scheme 1 Construction of two different structure types of complexes 1–2.

ACCEPTED MANUSCRIPT
Figure1.The monomer of Zn (II) in complex1

ACCEPTED MANUSCRIPT
Figure2.The 1D structure formed by the C–H…O hydrogen bonds in 1 (pink dashed lines present
the H-bond)

ACCEPTED MANUSCRIPT
Figure3.The monomer of Zn (II) in complex2

ACCEPTED MANUSCRIPT
Figure4. The 1D structure of the complex 2

ACCEPTED MANUSCRIPT
Figure5.Absorption spectral traces of complex 1in 5mM Tris-HCl–NaClbuffer (pH= 7.4) in the
absence (line 1) and presence (lines 2-8) of increasing amounts of CT-DNACDNA(lines
2-8):4,8,16,20,24,32,48umol/L

ACCEPTED MANUSCRIPT
Figure6.Absorption spectral traces of complex 2in 5mM Tris-HCl–NaClbuffer (pH= 7.4) in the
absence (line 1) and presence (lines 2-8) of increasing amounts of CT-DNACDNA(lines
2-8):4,8,16,20,24,32,48umol/L

ACCEPTED MANUSCRIPT
Figure7. Fluorescence spectra of the binding of EtBr to DNA in the absence(line 1) and
presence(line 2) of increasing amounts of the complexes λex = 526 nm, CEtBr = 1.0 ꢀmol/L, CDNA
= 5.1 ꢀmol/L, CM(1–4)(line 2-5): 2.5, 3.75, 6.25, 10 (ꢀmol/L)

ACCEPTED MANUSCRIPT
Figure8. Fluorescence spectra of the binding of EtBr to DNA in the absence (line 1) and presence
(line 2) of increasing amounts of the complexes λex = 526 nm, CEtBr= 1.0 ꢀmol/L, CDNA = 5.1
ꢀmol/L, CM(1–4)(line 2-5): 2.5, 3.75, 6.25, 10 (ꢀmol/L).

ACCEPTED MANUSCRIPT
Figure9.Cleavage of Pbr322 DNA (10 ꢀmol/L) in the presence of complex 1 and 2, Lane 0, DNA
alone; lanes 1–3, DNA + 2 (14.0, 21.0,28.0ꢀmol/L respectively); lanes 4–6, DNA + 1 (14.0,
21.0,28.0ꢀmol/L respectively)